One serotype-specific and three serotype-cross-reactive anti-VP7 neutralizing monoclonal antibodies (N-mAbs) were utilized to study the neutralization epitopes involved in the formation of antigenic sites on porcine rotavirus Gottfried strain (serotype 4). Single, double, or triple neutralization-resistant mutants were selected in the presence of these N-mAbs in vitro. Sequence analysis of the gene encoding VP7 of such mutants revealed that: (i) the amino acid (aa) that is substituted at a specific position on the mutant VP7 selected by a single N-mAb can vary, resulting in variants which exhibit antigenic differences; (ii) in addition to variable regions VR-5 (aa 87-100), VR-7 (aa 141-150, VR-8 (aa 208-224), and VR-9 (aa 235-242), aa 290 and 291 in the constant region of the VP7 are involved in neutralization, and (iii) VR-5, VR-7, VR-8, VR-9, and aa 290 and 291 are functionally related to one another. In order to further analyze neutralization sites on the VP7 and to study possible synergistic or antagonistic effects among the N-mAbs employed in this study, antigenic variants were generated in the presence of three N-mAbs. Nucleotide sequence analysis of the VP7-encoding gene of the mutants failed to demonstrate synergistic or antagonistic effects among the mAbs used in the selection of neutralization escape mutants.